Project Summary/Abstract Innate immune signaling by macrophages is critical as the first response to pathogen exposure. Three Interferon-regulatory factors (IRFs) are the critical regulators of the innate immune response (namely IRF3, ISGF3, and IRF7), and they respond to pathogen exposure coordinately and interdependently. How these IRFs function as a regulatory system, and what their specific roles are in regulating the extensive gene expression programs comprising hundreds of genes, has remained unclear, as previous assays lacked resolution to quantitate IRF dynamics at single cell resolution, and interdependencies between these factors could not be delineated by prevailing models. Thus the scientific premise of the proposed studies is that utilizing cutting-edge quantitative single cell and genome-wide measurements will allow us develop a quantitative mathematical model that delineates respective functions of IRF3, ISGF3, IRF7. We will address this goal with the following Aims: In Aim 1, we will elucidate the mechanism that are able to produce the complex dynamic control of the primary response factor IRF3 that we reveal in our preliminary studies. In Aim 2, we will investigate the functional consequence of differential, pathogen-specific IRF3 dynamics on chromatin control and target gene expression. In Aim 3, we will develop a predictive signaling model of the dose response and dynamic control of ISGF3 activated via autocrine and paracrine type I interferon secretion, and characterize how it contributes to innate immune gene expression in both infected and in bystander cells. In Aim 4, we will examine the regulatory control of IRF7 and test the hypothesis that it plays a key role in determining innate immune responses in previously interferon-`warned' cells, thus mediating a form of innate immune memory.